The Wiskott-Aldrich syndrome, a primary immunodeficiency in which the cytoskeletal integrity of hematopoietic cells is affected, is caused by a mutation in the WASP gene. We have identified a gene encoding a novel WASP Interacting Protein (WIP). In resting cells, WIP forms a complex with WASP/NWASP and inhibits the induction of their actin nucleating activity by Cdc42-GTP. We recently generated WIP-/- mice. Although WIP-/- T lymphocytes develop normally, they fail to proliferate, secrete IL-2, increase their F-actin content, or extend protrusions following T cell receptor ligation. Furthermore, they are deficient in contact formation with anti-CD3/I-CAM1-containing lipid bilayers and with B cells presenting superantigen. Consistent with a key role for WIP in actin reorganization, WIP-/- T cells display a profound defect in their ability to assemble subcortical actin filament networks. Our preliminary data suggests that WIP binds to the adapter protein CrkL that ZAP-70 recruits a CrkL/WIP-WASP complex to the TCR following TCR engagement, and that WIP is subsequently phosphorylated by PKCtheta and disengages from WASP. Our hypothesis is that a CrkL/WIP/WASP complex is recruited to the TCR in lipid rafts where WIP is phosphorylated by PKCtheta. This releases WASP from WIP, making it available for activation by Cdc42-GTP to initiate localized actin polymerization, which is essential for raft stability and for efficient concentration and integration of signaling molecules in the T cell immunological synapse (IS). The outcome is sustained activation of transcription factors that lead to optimal IL-2 gene expression and T cell proliferation. We propose to test this three step mechanistic model of WIP function in T cells by: 1) Analyzing the ability of WIP-/- T cells to form an IS, in which key signaling molecules are concentrated in lipids rafts leading to activation of the IL-2 gene. We will examine IS formation with anti-CD3 coated beads, MHC class II-peptide/ICAM-1 bilayers and antigen presenting cells; composition and stability of lipids rafts; and sustained activation of Ca++ mobilization, signaling intermediates and transcription factors that regulate IL-2 gene expression in WIP-/- T cells. 2) Dissecting the role of WIP domains that lack actin binding, CrkL binding, or WASP binding sites in TCR signaling. We will examine TCR signaling in WIP-/- T cells retrovirally reconstituted in vitro with WIP mutants, in T cells from RAG2-/- mice reconstituted with WIP-/- hemopoietic stem cells bearing WIP mutant transgenes and in T cells from WIP-/- mice reconstituted with mutant WIP transgenes. 3) Examining the role of CrkL in the recruitment of WIP and WASP to the TCR and in TCR signaling. We will examine the recruitment of the WIP-WASP complex in SLP-76 deficient T cells and in T cells that express dominant negative CrkL mutants and we will examine TCR signaling in CrkL-/- T cells. 4) Testing the hypothesis that phosphorylation of WIP by PKCtheta leads to the dissociation of WASP from WIP and its release from inhibition. We will study actin-based cytoskeletal changes following TCR ligation in PKCtheta-/- mice, the identity of key phosphorylated residue(s) in WIP that perturb WASP binding, and examine the effect of WIP phosphorylation on Cdc42-GTP driven activation of WASP. The results obtained will help clarify the link between the TCR and the actin cytoskeleton, will provide a better understanding of cell activation and potential important applications for immunodeficiency diseases, autoimmunity and cancer.